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Objectives:To establish a model of Mycobacterium tuberculosis infection of osteoclasts(OC)and explore the mechanism of Mycobacterium tuberculosis infection on OC.Methods:Peripheral blood mononuclear cells(peripheral blood mononuclear cells,PBMCs)were isolated from healthy volunteers.Receptor activator of nuclear factor-KB ligand(RANKL)and macrophage-colony stimulating factor(M-CSF)were used to make PBMCS into OC,and tartrate resistant acid phosphatase(TRAP)staining was performed on the cells.The constructed kanamycin resistant H37Rv pMV261-GFP green fluorescent strain was resuscitated and cultured with 10％oleic albumin dextrose catalase(OADC),7H9 and kanamycin containing Mycobacterium tuberculosis special liquid medium in an incubator at 37℃ until the optical density(OD)value was about 0.5 at 600nm.The OC cells cultured alone were set as the blank control group.And OC cells were also infected with Mycobacterium tuberculosis at different multiplicity of infection(MOI)for 24h,and MTT colorimetric method was used to detect cell survival rate.The MOI with the highest cell survival rate was selected as experimental MOI,and OC cells infected with H37Rv at experimental MOI were set as the experimental group.Fluorescence microscopy and Mycobacterium tuberculosis acid-fast staining were used to observe the transfection of Mycobacterium tuberculosis at the experimental MOI.Quantitative real-time PCR(qRT-PCR)was used to detect the expressions of non-receptor tyrosine kinase C-src,cathepsin K(CK),carbonic anhydrase 2(CA2),Integrin-β3 and matrix metalloproteinase-9(MMP-9).Immunohistochemistry was used to detect the expressions of P-src,CK,CA2,Integrin-β3 and MMP-9 on the cell surface.Western blot(WB)was used to detect the protein expression levels of P-src,CK,CA2,Integrin-β3,and MMP-9.Results:TRAP staining showed that more than 90％of the cells were OC after 15d of culture,which could be used for experiments.The results of MTT colorimetric assay showed that the cell survival rate was the highest when the MOI was 20:1(P＜0.05).This transfection multiplicity can be used as the concentration of experimental group.Fluorescence microscopy showed that when the transfection multiplicity ratio was 20:1,the green fluorescent Mycobacterium tuberculosis entered the OC and was successfully transfected into the OC.The results of acid-fast staining after infection of OC with Mycobacterium tuberculosis showed that when the MOI was 20:1,the acid-fast Mycobacterium tuberculosis stained red entered OC and was also successfully transfected into OC.The results of qRT-PCR,cell immunohistochemistry,and WB showed that the expressions of MMP-9,CK,C-src,CA2,and Integrin-β3 in the experimental group were higher than those in the blank control group(P＜0.05).Conclusions:Mycobacterium tuberculosis can transfect OC;Compared with the blank control group,the levels of five bone destruction factors in the experimental group transfected with OC by Mycobacterium tuberculosis were increased,suggesting that bone destruction of spinal tuberculosis may be related to this,which may provide a new exploration direction for the diagnosis and treatment of bone tuberculosis diseases.
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Objective: To investigate the effect of HOXD3 expression on the stem cell-like characteristics of breast cancer cells and the relationship between HOXD3 expression and multi-drug resistance in breast cancer cells. Methods: From January 2006 to December 2008, 87 specimens of breast cancer patients from the Affiliated Tumor Hospital of Harbin Medical University were collected. The ex-pression of HOXD3 in breast cancer cells and tissues was detected by immunohistochemical staining method. The expression levels of HOXD3 in CDDP or DOX-resistant cell lines MDA-MB-231 and MDA-MB-435 were detected by RT-PCR, Western blot and immunofluo-rescence staining. The effect of HOXD3 overexpression on the expression levels of stem cell biomarkers in breast cancer cell lines MDA-MB-231 and MDA-MB-435 was analyzed. MTT assay and colony formation assay were used to demonstrate the role of HOXD3 in che-motherapy resistance of breast cancer cells. Results: The relative expression of HOXD3 mRNA in breast cancer was 4.16, which was sig-nificantly higher than 2.05 in normal tissues adjacent to cancer; the relative expression levels of HOXD3 mRNA in breast cancer cell lines MDA-MB-231, MDA-MBB-435 and MCF-7 were 3.25, 2.84 and 2.23, which were all higher than 1.00 in normal breast epithelial cell line MCF-10A ( all P＜0.05 ). The IC50s of MDA-MB-231 and MDA-MB-435 cell lines resistant to CDDP or DOX were (20.82±0.05) μmol/L, (19.69±0.47) μmol/L, (32.26±0.23) mmol/L and (26.08±0.55) mmol/L, respectively. Both were higher than the corresponding original cell lines (all P＜0.05), and the drug resistance times were 2.47 and 3.10 or 1.86 and 2.08, respectively. The number of tumor spheres and stem cell biomarker expression levels of MDA-MB-231 and MDA-MB-435 with HOXD3 overexpression were significantly in-creased (all P＜0.05). Conclusions: The expression of HOXD3 plays an important role in the maintenance of stem cell-like properties and drug resistance of breast cancer cells. The results of this study will help us better understand the complexity of breast cancer and pro-vide a theoretical basis for the development of targeted molecular therapy.
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Objective To investigate the correlations of P2Y1 and ITGB3 single nucleotide polymorphisms (SNP) with aspirin resistance (AR) in patients with large atherosclerotic stroke (LAA) in a Chinese Han population.Methods Patients with first-ever LAA from Anhui stroke registration system were enrolled.Thrombus elasticity diagram was used to detect the platelet function.TaqMan technology was used to detect the P2Y1 and ITGB3 genotypes.Results A total of 206 patients with LAA were enrolled.Thirty-one patients (15.0％) had AR and 175 (85.0％) were aspirin sensitive (AS).The frequency of P2Y1 rs701265 G allele in the AR group was significantly higher than that in the AS group (43.5％ vs.26.9％;x2 =7.074,P=0.008).The frequency of P2Y1 rs701265 AA genotype in the AR group was significantly lower than that in the AS group (32.3％ vs.53.7％;x2 =4.850,P=0.028).There were no significant significances in the frequencies of P2Y1 rs1065776 and ITGB3 rs5918 alleles and genotypes between the AR group and the AS group.Multivariate logistic regression analysis showed that P2Y1 rs701265 G allele was an independent risk factor for AR in patients with LAA (odds ratio 2.186,95％ confidence interval 1.190-4.016;P=0.012).Conclusion The P2Y1 rs701265 polymorphism is associated with AR in Chinese Han patients with LAA,while the P2Y1 rs1065776 and ITGB3 rs5918 polymorphisms are not.
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Objective To study the effect of pioglitazone on the differentiation and function of rat osteoclast-like cells (OLC), and to probe the relationship between activated PPARγ2 and osteoclasts. Methods On day 1 of OLC formation from nonadherent bone marrow ceils (BMC) obtained from rats induced by M-CSF and receptor activator of NF-кB ligand (RANKL), 1, 5 and 10μmol/L pioglitazone hydrochloride was added. RT- PCR was performed to determine the mRNA expressions of PPARγ2 and receptor activator of NF-кB (RANK) on day 3, 5 and 7 during incubation, the number of tartrate-resistant acid phosphatase (TRAP)-positive cells,the number of bone resorption pits and the ratio of its area on dentin slice were counted, the activity of TRAP and the mean fluorescence intensity of integrin β3 (CD61) of OLC were also measured. Results (1) The effect on the differentiation of OLC: The addition of pioglitazone at the start of the culture period induced a dose-dependent decrease in TRAP-positive OLC and the activity of TRAP (P < 0.01 or P < 0.05) ; the mRNA expression of PPARγ2 was up-regulated by 5 and 10 μmol/L pioglitazone in the early stage of incubation and attenuated with thematuration of OLC on the contrary, however, the expression of RANK was down-regulated by 5 and 10 μmol/L piolitazone in every stage of incubation (P < 0.05 or P < 0.01), combined with decrease in TRAP-positive OLC from day 3 by 10 μmol/L pioglitazone. (2) The effect on the function of OLC: the number of bone resorption pits and the ratio of its area on dentin slice were decreased in groups of 5 and 10 μmol/L pioglitazone (P < 0.01 orP < 0.05), no obvious change was noted in the group with 1 μmol/L pioglitazone compared with the control group; the mean fluorescence intensity of CD61 were down-regulated in groups of 5 and 10 μmol/L pioglitazone (P < 0.05 or P <0.01). Conclusion Activation of PPARγ2 pathway by pioglitazone could partially inhibit differentiation and function of OLC derived from rat BMC.